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Lysis, binding and/or wash reagent for isolating and/or purifying nucleic acids

a technology of nucleic acid and lysis buffer, which is applied in the direction of microorganism lysis, biochemistry apparatus and processes, dna preparation, etc., can solve the problems of reducing the yield of isolated nucleic acids and the stability of lysis buffers containing polyoxyethylene sorbitan monolaurate (tween® 20)

Active Publication Date: 2017-04-11
QIAGEN GMBH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The reagent maintains stable pH and reduces contamination, resulting in improved nucleic acid yield and clarity of eluates even after storage, eliminating the need for additional purification steps.

Problems solved by technology

Disadvantageously, lysis buffers containing polyoxyethylene sorbitan monolaurate (Tween® 20) are not stable during storage.
A particular disadvantage is the fact that the yield of the isolated nucleic acids is reduced after storage when these lysis buffers are used.
Another disadvantage is the fact that the eluates containing nucleic acids are opaque, indicating the presence of contaminations which may interfere with further usage of the isolated nucleic acids.
Advantages arise in particular from the achievable good yields, even after storage of the lysis and / or binding reagents.

Method used

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  • Lysis, binding and/or wash reagent for isolating and/or purifying nucleic acids
  • Lysis, binding and/or wash reagent for isolating and/or purifying nucleic acids

Examples

Experimental program
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example 2

Extraction of Viral DNA

[0170]Negative, i.e. HBV virus-free, human plasma was admixed with 104 sgU / ml hepatitis B virus (HBV). The viral DNA was extracted from in each case 1000 μl of the plasma sample, using the commercially available automation platform QIAsymphony® (Qiagen), by means of the automated protocol for purifying viral nucleic acids from plasma samples.

[0171]According to the protocol used, the sample was exposed to the protocol-defined volumes—of lysis reagent B containing guanidinium isothiocyanate, tris(hydroxymethyl)aminomethane and 20% (w / v) Brij® 58 (Sigma) and proteinase K, and solution AVE containing carrier RNA. This was followed by incubation at 65° C. to lyse the sample. To the sample mix was then added the protocol-defined volume of binding reagent C containing guanidinium isothiocyanate, tris(hydroxymethyl)aminomethane and 9% (w / v) Brij® 58 (Sigma), and isopropanol. After a further 3 minutes of incubation, MagAttract suspension which contained the magnetic si...

example 3

Extraction of Viral DNA After Storage of the Lysis Reagent

[0174]The lysis reagents B containing guanidinium isothiocyanate, tris(hydroxymethyl)aminomethane and 20% (w / v) Brij® 58 (Sigma), and A in which Brij® 58 had been replaced with 20% (w / v) Tween® 20 (Fluka) were each stored in sealed vessels at 50° C. for 10 weeks.

[0175]The viral nucleic acid was then extracted, using the commercially available automation platform QIAsymphony® (Qiagen), by means of the automated protocol for purifying viral nucleic acid from plasma samples.

[0176]According to the protocol described in Example 2, the viral DNA was extracted from in each case 1000 μl of the plasma sample using the commercially available automation platform QIAsymphony® (Qiagen), with, for different mixtures, lysis reagent B containing guanidinium isothiocyanate, tris(hydroxymethyl)aminomethane and 20% (w / v) Brij® 58 (Sigma), and lysis reagent A in which Brij® 58 had been replaced with 20% (w / v) Tween® 20 (Fluka) being stored in ea...

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Abstract

The present invention relates to a lysis, binding and / or wash reagent for isolating and / or purifying nucleic acids and a method for isolating and / or purifying nucleic acids.

Description

FIELD OF THE INVENTION[0001]The present invention relates to a lysis, binding and / or wash reagent and to a method for isolating and / or purifying nucleic acids. Said lysis, binding and / or wash reagent and method are particularly suitable for use purposes in molecular diagnostics.BACKGROUND OF THE INVENTION[0002]The prior art has disclosed a multiplicity of methods for isolating and / or purifying nucleic acids such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) from cells, cell cultures or virus cultures.[0003]In this context, “classical” methods for isolating nucleic acids, many of which are carried out manually, are based on a one-step method which involves carrying out an extraction after the addition of an aqueous buffer and an organic extractant. The nucleic acids remain in the aqueous phase and may be isolated after the organic phase which contains undesired accompanying substances has been removed.[0004]These methods firstly use normally harmful organic extractants suc...

Claims

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Application Information

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Patent Type & Authority Patents(United States)
IPC IPC(8): C12N15/10
CPCC12N15/1003C12N15/1013C12N1/06C12Q1/6806C12N15/1006
Inventor FABIS, ROLANDHOMANN-WISCHINSKI, ANKEVOSS, THORSTENHANSELLE, THOMAS
Owner QIAGEN GMBH